自噬在熊果酸诱导的人脐静脉内皮细胞损伤中的作用

目的: 研究自噬在熊果酸诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤中的作用。方法: 体外培养HUVECs,运用不同浓度的熊果酸处理36 h,采用MTT法检测HUVECs增殖率的变化;透射电镜观察细胞超微结构的改变;单丹磺酰尸胺(MDC)荧光染色观察熊果酸对HUVECs自噬表达水平的影响;Western blotting检测微管相关蛋白轻链3(microtubule-associated protein light chain 3, LC3)及自噬相关蛋白Beclin-1的变化;RT-PCR检测LC3及Beclin-1 mRNA的变化;流式细胞术检测自噬抑制剂3-甲基腺嘌呤(3-methyladenine, 3-MA)抑制自噬前后经熊果酸处理的HUVECs凋亡率的变化。结果: 熊果酸抑制HUVECs增殖并呈剂量依赖性;熊果酸可诱导HUVECs产生自噬:透射电镜和MDC染色均显示HUVECs经熊果酸处理后自噬囊泡明显增加且自噬在基因及蛋白水平的表达均增加;3-MA与熊果酸联合作用于HUVECs时抑制了熊果酸诱导的HUVECs自噬空泡的积聚并且可明显促进HUVECs凋亡。结论: 熊果酸抑制HUVECs增殖,并诱导其发生自噬,自噬在此过程中对HUVECs起保护作用。自噬抑制剂3-MA能够增强熊果酸诱导的HUVECs的增殖抑制作用并且促进HUVECs发生凋亡。

Role of autophagy in ursolic acid-induced injury of human umbilical vein endothelial cells

AIM: To investigate the role of autophagy in the injury of human umbilical vein endothelial cells(HUVECs) induced by ursolic acid(UA).METHODS: HUVECs were cultured in vitro with UA at various concentrations for 36 h and the proliferation inhibitory rate of HUVECs was determined by MTT method.The change of ultrastructure was observed under transmission electronic microscope(TEM).The autophagy was observed using fluorescent microscope by monodansylcadaverin(MDC) staining.The protein level and mRNA expression of microtubule-associated protein light chain 3(LC3) and Beclin-1 were detected by Western blotting and RT-PCR,respectively.Cell apoptotic rate was measured by flow cytometry analysis.RESULTS: UA at various concentrations showed significantly dose-dependent inhibitory effect on the proliferation of HUVECs.Autophagy was induced in HUVECs treated with UA as detected by MDC staining and TEM.The protein level and mRNA expression of LC3 and Beclin-1 in HUVECs were significantly increased following the treatment with UA,which was also in a time-dependent manner.Compared with UA group,addition of 3-methyladenine(3-MA) inhibited the increase in autophagic vacuoles and exacerbated the apoptosis.CONCLUSION: Autophagy shows protective effect on the proliferation inhibition of HUVECs induced by UA and the proliferation inhibition can be enhanced by the autophagy inhibitor 3-MA.3-MA may enhance the apoptotic rate of HUVECs induced by UA.